We reported earlier that genetic deletion of TNF in resistant C57BL/6 mice (B6.TNF-/-) rendered them susceptible to fatal herpes simplex virus type 1 (HSV) infection. In further studies, we showed that B6 mice genetically deleted for either or both of the known TNF receptors TNFR1 or p55 and TNFR2, or p75 were resistant to fatal HSV infection. This surprising result suggested that TNF mediated resistance to HSV infection was mediated by a receptor distinct from the known TNF p55 (TNFR1) and p75 (TNFR2) receptors. We confirmed that TNF signaling mediated protection in mice by showing that Treatment of HSV infected double knockout B6.p55-/-p75-/- (dKO) mice with either a soluble TNFR1 preparation that binds only TNF and not lymphotoxin- (LT-)or a monoclonal antibody (mAb) targeting TNF increased mortality to the same extent, including in wild type (wt) control B6 mice which confirms the protective role of TNF signaling in dKO mice. Both TNF and its receptors exist as both membrane bound and as soluble proteins, hence a caveat with these approaches is that we could not exclude the possibility of reverse signaling through membrane TNF initiated by sTNFR and/or the anti-TNF mAb somehow impaired resistance. We therefore devised an in vivo siRNA approach to specifically down regulate TNF without affecting LT-. In vivo siTNF treatment increased mortality to the same extent in HSV infected dKO and B6 wt mice while the scrambled control siTNF had no effect. Thus, TNF protection against lethal HSV infection in dKO mice is mediated by signaling via a novel TNF receptor. In support of this hypothesis we present extensive preliminary data demonstrating specific binding of TNF conjugated to Alexa 488 or biotin (Alexa-TNF/Bio) to various immune cell types in dKO mice in a pattern distinct from that observed for wt and p55-/- B6 mice. We propose using a bifunctional crosslinker coupled to TNF to crosslink the new TNFR (nTNFR) on peritoneal macrophages from dKO mice. The purified TNF-nTNFR complex will be enzymatically fragmented and the identity of nTNFR will be established by liquid chromatography - tandem mass spectrometry (LC- MS/MS) analysis of the resulting tryptic peptides. nTNFR will characterized in term of its pattern of expression, ligand binding including LT-a and Progranulin, the newly identified ligand for p75 (TNFR2), structural relationship to p55 and p75, conservation in humans and role in host defense, inflammatory responses and autoimmunity. Results from these studies will significantly enhance understanding TNF signaling in the context of innate immunity, in various autoimmune diseases in which TNF plays a central role as well as in regulation of inflammation.